1. Field of the Invention
This invention relates to polyamides which bind to pre-determined sites of the minor groove of double-stranded DNA.
2. Description of the Related Art
The art describes a large variety of polyamides which have three to six carboxamide base pairs and a hairpin loop derived from γ-aminobutyric acid and the ability to bind to the minor groove of DNA in the promoter region to inhibit gene expression. Thus, polyamides consisting of N-methylimidazole (Im), N-methylpyrrole (Py), and β-alanine and γ-amino butyric acid and methods for preparation of such polyamides are well known.
Polyamides containing N-methylpyrrole and N-methylimidazole amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to naturally occurring DNA binding proteins (Trauger, et al. Nature 1996, 382, 559; Swalley, et al. J. Am. Chem. Soc. 1997, 119, 6953; Turner, et al. J. Am. Chem. Soc. 1997, 119, 7636). DNA recognition depends on side-by-side amino acid pairings oriented N-C with respect to the 5′-3′ direction of the DNA helix in the minor groove (Wade, W. S., et al. J. Am. Chem. Soc. 1992, 114, 8783; Mrksich, et al. Proc. Natl. Acad. Sci., USA 1992, 89, 7586; Wade, et al. Biochemistry 1993, 32, 11385; Mrksich, et al. J. Am. Chem. Soc. 1993, 115, 2572; Geierstanger, et al. Science 1994, 266, 646; White, et al. J. Am. Chem. Soc. 1997, 119, 8756). Antiparallel pairing of imidazole (Im) opposite pyrrole (Py) recognizes a G•C base pair, while a Py/Im combination recognizes C•G.2 A Py/Py pair is degenerate and recognizes either an A•T or T•A base pair (Wade, W. S., et al. J. Am. Chem. Soc. 1992, 114, 8783; Mrksich, et al. Proc. Natl. Acad. Sci., USA 1992, 89, 7586; Wade, et al. Biochemistry 1993, 32, 11385; Mrksich et al. J. Am. Chem. Soc. 1993, 115, 2572; Geierstanger, et al. Science 1994, 266, 646; White, et al. J. Am. Chem. Soc. 1997, 119, 8756; Pelton, et al. Proc. Natl. Acad. Sci., USA 1989, 86, 5723; Pelton, et al. J. Am. Chem. Soc. 1990, 112, 1393; White, et al. Biochemistry 1996, 35, 12532; Chen, et al. J. Mol. Biol. 1997, 267, 1157). An Im/Im pairing is disfavored, breaking a potential degeneracy for recognition (Singh, et al. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7673; White, et al. Chem. & Biol. 1997, 4, 569).
Investigators have also attempted to prevent slipped-binding motifs as well as increase DNA-binding affinity and sequence specificity by covalent linkage of polyamide subunits (Trauger, et al. J. Am. Chem. Soc. 1996, 118, 6160; Geierstanger, et al. Nature Struct. Biol. 1996, 3, 321; Swalley, et al. Chem. Eur. J. 1997, 3, 1608; Wemmer, et al. Curr. Opin. Struct. Biol. 1997, 7, 355; Mrksich, et al. J. Am. Chem. Soc. 1994, 116, 3663; Dwyer, et al. J. Am. Chem. Soc. 1993, 115, 9900; Chen, et al. J. Am. Chem. Soc. 1994, 116, 6995). A hairpin polyamide motif with γ-aminobutyric acid (γ) has been utilized as a turn-specific internal-guide-residue and provides a synthetically accessible method for C-N linkage of polyamide subunits (FIG. 1). Head-to-tail linked polyamides bind specifically to designated target sites with 100-fold enhanced affinity relative to unlinked subunits (Mrksich, et al. J. Am. Chem. Soc. 1994, 116, 7983; Parks, et al. J. Am. Chem. Soc. 1996, 118, 6147; Parks, et al. J. Am. Chem. Soc. 1996, 118, 6153; Trauger, et al. Chem. & Biol. 1996, 3, 369; Swalley, et al. J. Am. Chem. Soc. 1996, 118, 8198; Pilch, et al. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8306; de Claire, et al. J. Am. Chem. Soc. 1997, 119, 7909).
Eight-ring hairpin polyamides bearing a single positively charged tertiary amine group at the C-terminus have been shown to be cell-permeable and to inhibit the transcription of specific genes in cell culture (Gottesfeld, et al. Nature 1997, 387, 202). However, recent studies of polyamide size limitations suggest that beyond five rings, the ligand curvature fails to match the pitch of the DNA helix, disrupting the hydrogen bonds and van der Waals interactions responsible for specific polyamide-DNA complex formation (Kelley, et al. Proc. Natl. Acad. Sci. USA, 1996, 93:6981; Kielkopf, et al. Nature Struc. Biol., in press). Recognition of seven base pairs by ten-ring hairpin polyamids containing five contiguous ring pairings represents the upper limit in binding site sizes targetable by the hairpin motif (Turner, et al. J. am. Chem. Soc., 1997, 119:7636). Addition of pairings of β-alanine with β-alanine, pyrrole, or imidazole has allowed extention of the hairpin motif to 8-bp recognition, as demonstrated in provisional application 60/042,222. However, those skilled in the art have recognized the extreme difficulties associated with the design of hairpin motifs recognizing longer site sizes.
The present invention involves the use of R-2,4-diaminobutyric acid as a replacement for γ-aminobutyric acid to make the hairpin loop. In addition, a methodology for expanding the targetable binding site size of hairpins by covalently linking existing hairpin motifs without compromising DNA-binding and sequence specificity is provided.